Although the study subjects were seronegative to the hemagglutinins in the vaccine viruses and were predicted to be susceptible to infection with the pLAIVs because the HA and NA genes were derived from novel influenza virus subtypes from avian/animal species, the vaccine viruses were highly restricted in replication. The immune responses to the pLAIVs were variable, ranging from a high seroconversion rates to H9N2 and H7N3 to low seroconversion rates for H5N1, H6N1 and H2N2 vaccines. In order to pursue a live attenuated influenza vaccine strategy for influenza viruses with pandemic potential, it is important to learn more about factors that determine the level of replication of the pLAIV strains in seronegative healthy adults and factors that determine whether a vaccine recipient will develop an immune response to the vaccine. In FY13, we undertook a study to identify biomarkers that would that predict shedding and immune response to live attenuated influenza virus (LAIV) vaccines. This study would address the following questions: (1) Which biomarkers predict whether seasonal or pandemic LAIV replicate in the respiratory tract of healthy adults? (2) Which biomarkers predict whether seasonal or pandemic LAIV will elicit an immune response in healthy adults? These studies are in progress. We had previously generated two H5N1 pandemic live attenuated influenza vaccines (LAIV); unfortunately, few vaccinees shed virus and/or seroconverted by hemagglutination inhibition or neutralization. We sought to determine whether the vaccines primed or established long lasting immunity that could be detected by administration of inactivated H5N1 influenza vaccine (ISIV). Persons who previously received H5N1 pLAIV, persons who previously received an irrelevant H7N3 pLAIV, and H5N1- and LAIV-naive community members were invited to participate in the study. LAIV experienced subjects received a single 45microgram dose of H5N1 ISIV. H5N1-and LAIV-naive subjects received either one or two doses of ISIV. We observed an increased frequency (82% vs. 50%) and significantly higher titer (geometric mean antibody titer 112 vs. 76) hemagglutination inhibition antibody response (p=004) in subjects who had previously received antigenically matched H5N1 pLAIV followed by one dose of ISIV compared with H5N1-and LAIV-naive two dose ISIV recipients. The affinity of antibody was also enhanced in H5N1 pLAIV-primed subjects. We also evaluated the quality of the antibody response and found that priming with H5N1 pLAIV induced antibody with a greater breadth of reactivity against different clades of H5N1 influenza viruses than that observed in those individuals who only received ISIV. The high titer, rapid antibody response following a single dose of unadjuvanted H5N1 ISIV in a majority of subjects who had received an H5N1 pLAIV almost five years earlier is clear evidence that H5N1 pLAIV priming induced long lasting B cell memory. The magnitude and frequency of the neutralizing antibody response in recipients of the H5N1 pLAIVs is remarkable given the paucity of such responses in these participants after the priming doses of pLAIV, as well as the highly restricted replication of H5N1 pLAIV in these individuals. These results demonstrate the utility of using an inactivated vaccine to probe for the immune response generated by the pLAIV. Cross-reactivity is important because ten distinct clades of H5N1 influenza viruses have been identified in avian species since 2003. H5N1 viruses continue to evolve in nature and we do not know whether some clades pose a greater pandemic threat than others. Therefore, a vaccination strategy that induces a broadly cross-reactive antibody response is highly desirable. In summary, ISIV administration unmasked long lasting immunity in H5N1 pLAIV recipients with a rapid, high titer, high quality antibody response that was broadly cross-reactive across several H5N1 clades. The high titer and high quality, broadly cross-reactive antibody response seen in our study in which pLAIV priming was unmasked by an ISIV boost should raise interest in unconventional vaccination schedules that combine different types of vaccines. Future studies will be designed to determine how optimal priming can be achieved.